Employing relaxed stringency conditions in Southern blot hybridization experiments with v-erbB as a probe, we have previously identified and partially isolated a novel human v-erbB-related gene which was amplified in a primary mammary adenocarcinoma (mac) and was distinct from the EGF receptor gene. Molecular cloning of a 6-kb genomic Eco RI fragment of mac and nucleotide sequence analysis defined two putative exons with closer homology to v-erbB and human EGF receptor than to other reported tyrosine kinases. Using an exon-specific genomic mac probe, we identified a 4.8-kb specific mRNA in A431 cells distinct from the three major EGF receptor gene transcripts in this cell line. Expression of mac was observed in a broad spectrum of human tissues of both epithelial and mesodermal origin. In vitro autokinase activity was intrinsic to the 185,000 dalton protein product immunoprecipitated by a specific peptide antibody. In order to investigate the normal structure and potential alterations of mac in human mammary neoplasia, we isolated the entire coding region by cDNA cloning. Subsequent Southern and Northern blot analysis of human mammary tumors and tumor cell lines using cDNA probes revealed gene amplification of mac with overexpression in 6/62 cases. Moreover, we observed overexpression of apparently normal size mac mRNA without detectable gene amplification in 3/16 mammary tumor cell lines, suggesting a mac overexpression due to transcriptional dysregulation in these cases. In addition, using EGF receptor cDNA probes, 3/50 mammary tumors and tumor cell lines exhibited gene amplification with overexpression of an apparently normal size transcript.